scholarly journals Kinetics of Alkaline Phosphatase from Pig Kidney. Influence of complexing agents on stability and activity

1976 ◽  
Vol 153 (2) ◽  
pp. 151-157 ◽  
Author(s):  
B P Ackermann ◽  
J Ahlers
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ion ◽  
Complexing Agents ◽  
Pig Kidney ◽  
Low Concentrations ◽  
Sequential Addition ◽  
Quantitative Examination ◽  
High Concentrations ◽  
Kinetics Of ◽  
Active Centres

Metal ion-complexing agents, like KCN, EDTA etc., inactivate alkaline phosphatase of pig kidney. This inactivation is reversible at low concentrations of the complexing agents and irreversible at high concentrations. The reversible inhibition is probably due to removal of Zn2+ ions from the active site, where they are necessary for catalytic action, whereas the irreversible inhibition results from the removal of Zn2+ ions necessary for preservation of the structure. The inactivation is pseudo-first order. It depends on the concentration, size and charge of the complexing agents. β-Glycerophosphate and Mg2+ ions protect the enzyme from inactivation by complexing agents. Quantitative examination of the effect of substrate leads to a model that is similar to the “sequential model” proposed by D.E. Koshland, G. Nemethy & D. Filmer (1966) (Biochemistry 5, 365-385) to explain allosteric behavior of enzymes. It describes the sequential addition of two substrate molecules at two active centres of the dimer enzyme. The binding of the substrate molecules is accompanied by changes in the conformation, which lead to stabilization of the enzyme against attack by complexing agents.

scholarly journals The reversible inactivation of pig kidney alkaline phosphatase at low pH

1968 ◽  
Vol 108 (2) ◽  
pp. 243-246 ◽  
Author(s):  
P. J. Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Treatment ◽  
Gel Filtration ◽  
Reversible Inactivation ◽  
Pig Kidney ◽  
Reactivation Mechanism ◽  
Increasing Temperature ◽  
Filtration Properties ◽  
Kinetics Of ◽  
Kidney Alkaline Phosphatase

1. Pig kidney alkaline phosphatase is inactivated by treatment with acid at 0°. 2. Inactivated enzyme can be partially reactivated by incubation at 30° in neutral or alkaline buffer. The amount of reactivation that occurs depends on the degree of acid treatment; enzyme that has been inactivated below pH3·3 shows very little reactivation. 3. Studies of the kinetics of reactivation indicate that the process is greatly accelerated by increasing temperature and proceeds by a unimolecular mechanism. The reactivated enzyme has electrophoretic and gel-filtration properties identical with those of non-treated enzyme. 4. The results can be best explained by assuming that a lowering of the pH causes a reversible conformational change of the alkaline phosphatase molecule to a form that is no longer enzymically active but is very susceptible to permanent denaturation by prolonged acid treatment. A reactivation mechanism involving sub-unit recombination seems unlikely.

scholarly journals Kinetics of alkaline phosphatase from pig kidney. Mechanism of activation by magnesium ions

1974 ◽  
Vol 141 (1) ◽  
pp. 257-263 ◽  
Author(s):  
Jan Ahlers
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Metal Ion ◽  
Ion Concentration ◽  
British Library ◽  
Kinetic Measurements ◽  
Pig Kidney ◽  
Enzyme Substrate ◽  
Lending Library ◽  
Model Scheme

The mechanism of activation of alkaline phosphatase (EC 3.1.3.1) from pig kidney by Mg2+ ions was investigated with the aid of kinetic measurements. Mg2+ ions are essential for enzyme activity. The following model (Scheme 1 of the text) for the reaction of enzyme, substrate and Mg2+ ions was derived: [Formula: see text] The binding of the substrate to the enzyme is independent of the binding of the activator, and vice versa. Mg2+ must therefore play a part in the substrate decomposition. It is not possible to determine whether the Mg2+ ions are involved directly in the catalytic process, or whether they act as regulatory effectors. Because of the strong affinity existing between the alkaline phosphatase and Mg2+, it is necessary to adjust the metal-ion concentration with the aid of a metal buffer. In the Appendix the necessary equations are derived for calculating the concentration of free metal ions in a system with several different metal ions. A FORTRAN IV program for solving these equations and for graphic presentation of the results has been deposited as Supplementary Publication SUP 50030 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS 23 7 BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

scholarly journals Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase

1982 ◽  
Vol 205 (2) ◽  
pp. 443-448 ◽  
Author(s):  
F M Dickinson ◽  
G J Hart
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Metal Ion ◽  
Stopped Flow ◽  
Maximal Effect ◽  
Ion Concentrations ◽  
Sheep Liver ◽  
High Concentrations ◽  
Flow Experiments ◽  
Active Centres

Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.

scholarly journals Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1972 ◽  
Vol 127 (1) ◽  
pp. 271-283 ◽  
Author(s):  
R. G. Hansford
Keyword(s):  
Oxygen Uptake ◽  
Isocitrate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Slight Reduction ◽  
Glycerol Phosphate ◽  
Pyruvate Oxidation ◽  
Low Concentrations ◽  
High Concentrations ◽  
Kinetics Of ◽  
Very High

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl2 during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H+ ions, and inhibited by ATP, NADH and Ca2+. 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a `classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.

scholarly journals Steady-state kinetics of catecholamine transport by chromaffin-granule ‘ghosts’

1974 ◽  
Vol 144 (2) ◽  
pp. 319-325 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Affinity Constant ◽  
Chromaffin Granule ◽  
Affinity Constants ◽  
Steady State Kinetics ◽  
Low Concentrations ◽  
High Concentrations ◽  
Chromaffin Granule Ghosts ◽  
Kinetics Of

Resealed chromaffin-granule ‘ghosts’ were used to study the steady-state kinetics of catecholamine transport. The pump has a high affinity for (-)-noradrenaline, (-)-adrenaline, tyramine and 5-hydroxytryptamine (serotonin), but a lower affinity for (+)-noradrenaline. The measured rates of incorporation do not conform to Michaelis–Menten kinetics, but affinity constants for the former substrates are in the range 8–18μm. Reserpine is a potent inhibitor. Incorporation as a function of ATP concentration also fails to show simple kinetics; the affinity constant for ATP is deduced to be about 3mm at 1mm-MgCl2. Adenylyl (βγ-methylene)diphosphonate is a competitive inhibitor at low concentrations, but inhibits more strongly at high concentrations. The pump has a transition temperature at 29°C and does not seem to be identical with the Mg2+-stimulated adenosine triphosphatase of chromaffin granules.

Kinetics of Uptake of Lysine and Lysyl-Lysine by Hamster Jejunum in Vitro

1980 ◽  
Vol 59 (4) ◽  
pp. 285-287 ◽  
Author(s):  
D. Burston ◽  
E. Taylor ◽  
D. M. Matthews
Keyword(s):  
Amino Acid ◽  
Molar Basis ◽  
Low Concentrations ◽  
High Concentrations ◽  
Kinetics Of Uptake ◽  
Kinetics Of

1. The kinetics of 2-min uptake of l-lysine and l-lysyl-l-lysine have been studied by using rings of everted hamster intestine in vitro, and values for Kt and Vmax, established. 2. On a molar basis, mediated uptake was more rapid for the amino acid than for the peptide. Non-mediated uptake was more rapid for the peptide than for the amino acid. 3. Comparison of relative rates of uptake of lysine from equivalent solutions of lysine and lysyl-lysine showed that at low concentrations, uptake of lysine was less rapid from the peptide than from the amino acid, whereas at high concentrations, uptake of lysine was more rapid from the peptide than from the amino acid. This type of effect of concentration on relative rates of uptake from equivalent solutions of amino acid and peptide has not previously been described.

scholarly journals Kinetics of substrate hydrolysis and inhibition by mipafox of paraoxon-preinhibited hen brain esterase activity

1986 ◽  
Vol 236 (2) ◽  
pp. 503-507 ◽  
Author(s):  
C D Carrington ◽  
M B Abou-Donia
Keyword(s):  
Kinetic Parameters ◽  
Organophosphorus Compounds ◽  
Model Fit ◽  
Component Model ◽  
Low Concentrations ◽  
Two Component ◽  
High Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Organophosphorus Inhibitor

For the purpose of assessing the neurotoxic potential of organophosphorus compounds, it has been determined that paraoxon-preinhibited hen brain has both neurotoxicant (mipafox)-sensitive (neurotoxic esterase; NTE) and -insensitive esterase components. Several experiments designed to investigate the kinetic parameters governing the reaction of these esterases with two substrates and one organophosphorus inhibitor are presented. First, kinetic parameters for the hydrolysis of phenyl valerate and phenyl phenylacetate were measured. At 37 degrees C, the Km values of NTE for phenyl valerate and phenyl phenylacetate were found to be about 1.4 × 10(-3) and 1.6 × 10(-4) M respectively. At 25 degrees C, the Km of NTE for phenyl valerate was determined to be about 2.4 × 10(-3) M. Secondly, the kinetic constants of NTE for mipafox were measured at both 25 degrees C and 37 degrees C. With either phenyl valerate or phenyl phenylacetate as substrate, the Km at 37 degrees C was determined to be about 1.8 × 10(-4) M, and the phosphorylation constant (k2) was about 1.1 min-1. For phenyl valerate only, the Km at 25 degrees C was found to be about 6 × 10(-4) M, and the k2 was about 0.7 min-1. The data obtained at 25 degrees C were analysed by using a two-component model without formation of Michaelis complex, a two-component model with formation of Michaelis complex on the second component (NTE), or a three-component model without formation of Michaelis complex. The fact that the Michaelis model fit the data significantly better than either of the other two models indicates that the higher apparent Ki values that occur with low concentrations of mipafox are due to formation of Michaelis complex at high concentrations, rather than because of the presence of two NTE isoenzymes, as has been suggested by other investigators.

scholarly journals The non-enzymic hydroxylation of phenylalanine to tyrosine by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine

1971 ◽  
Vol 125 (2) ◽  
pp. 569-574 ◽  
Author(s):  
L. I. Woolf ◽  
A. Jakubovič ◽  
E. Chan-Henry
Keyword(s):  
Chromatographic Separation ◽  
Phenylalanine Hydroxylase ◽  
Complexing Agents ◽  
Thiol Compounds ◽  
Enzymic Reaction ◽  
Low Concentrations ◽  
High Concentrations ◽  
Metal Complexing Agents

1. Phenylalanine is converted into tyrosine by incubation in air with 6,7-dimethyltetrahydropterin, which is a cofactor for the enzymic hydroxylation. This can cause serious inaccuracies in assays of phenylalanine hydroxylase. 2. The non-enzymic reaction is not specific for l-phenylalanine. 3. m-Tyrosine, o-tyrosine and dihydroxyphenylalanines are formed in addition to p-tyrosine; their chromatographic separation and assay are described. 4. l-[14C]Phenylalanine as purchased or soon after purification contains p- and m-tyrosine, both of which can cause errors in the assay of phenylalanine hydroxylase. 5. Catalase prevents the non-enzymic hydroxylation. Thiol compounds in low concentrations stimulate the reaction but in high concentrations are inhibitory. Fe2+ and metal complexing agents have small stimulatory effects. 6. The mechanism of the non-enzymic reaction and its possible relation to the enzymic hydroxylation of phenylalanine are discussed; it is suggested that phenylalanine is attacked by a peroxide of the cofactor.

Sign in / Sign up

Export Citation Format

Share Document